Use of Caenorhabditis elegans as a model to study Alzheimer’s disease and other neurodegenerative diseases

1Alzheimer’s disease (AD) is the 6th leading cause of death in the US and affects more than 35 million people worldwide. AD is a neurodegenerative disease characterized by a progressive loss of memory. Most cases of AD occur sporadically in aged people (>60 years, late-onset AD) without a clear inheritance pattern. However, in 5% of the cases (familial or early onset AD) AD symptoms appear earlier and are linked with gene mutations. Both forms of AD have two main neuropathologic features: the presence of extra-neuronal amyloid plaques, often referred to as senile plaques, and intraneuronal neurofibrillary tangles. Amyloid plaques are aggregates of the beta-amyloid peptide (Aβ), a cleavage product of the amyloid precursor protein (APP;;; ). Hyperphosphorylation of the microtubule associated protein tau causes its polymerization into paired helical filaments (PHFs) and, presumably, its formation into neurofibrillary tangles.

2Mutations in the APP gene and/or the enzymes involved in APP processing (γ-secretase components presenilins, PSEN1 and PSEN2;;;;;; ) are correlated with early onset AD. These mutations increase the levels of toxic Aβ species and promote neurodegeneration. By contrast, a recently identified mutation in APP affects cleavage of APP, causing less Aβ production and conferring neuroprotective benefits. Despite the significant advances made using APP transgenic and knockout models in mammals, unraveling the cellular role of APP has been difficult. Alternative animal models provide complementary approaches to dissecting the function of APP and tau. In this review, we discuss the latest uses of the nematode Caenorhabditis elegans as a model system for the study of AD. We also include a brief review of a few representative examples of how C. elegans is being utilized to model other neurodegenerative diseases.

3Caenorhabditis elegans is a free-living, non-parasitic nematode that was first introduced as a model organism by Sydney Brenner in 1963. It is a small (1 mm in length), transparent roundworm, which makes it easy for manipulation, and has a short life cycle of 3 days from egg to adult at 25°C. Under suitable growing conditions, hatched animals develop through four larval stages (L1–L4), each punctuated by a molt, to arise as an adult hermaphrodite with 959 somatic cells. Its life span is between 2 and 3 weeks, which facilitates the study of its biology. Completion of the C. elegans genome sequence in 1998 demonstrated that roughly 38% of worm genes have a human ortholog, such as APP and tau. Hence, C. elegans has many excellent advantages as an in vivo model for the study of AD and other neurodegenerative diseases.

4The APP family of proteins contains three members, APP, APLP1, and APLP2, which are characterized by a large extracellular region containing conserved E1 and E2 domains, a single transmembrane domain, and a small cytosolic domain. APLP1 and APLP2 do not contain the Aβ sequence and, hence, do not produce Aβ (Figure 1A;, ). The APP gene family is required for viability and brain development. APP mouse knockouts were viable, but had behavioral and cognitive defects. While knockout of APLP1 resulted in postnatal growth defects, mice in which APLP2 was inactivated appeared wild type. Double knockouts of APLP2 and either APP or APLP1, however, resulted in postnatal lethality; the lethality of APP/APLP2 double knockouts could be rescued by knock-in of an APP extracellular fragment, sAPPα, suggesting that sAPPα is sufficient for viability. The triple knockout caused lethality and a type II lissencephaly and cortical disorganization. Collectively, these results suggest that APP family members have essential and redundant functions during development, including proper brain development, and these functions do not require Aβ.

5In mammals, APP is processed through two proteolytic pathways, only one of which produces Aβ (Figure 1B;,,). In the non-amyloidogenic pathway, APP is first cleaved by an α-secretase within the Aβ sequence to release an extracellular fragment, sAPPα (Figure 1B). The remaining APP fragment (known as APP-CTFα or C83) is then cleaved by the γ-secretase complex to release the APP intracellular domain (AICD) to the cytosol. By contrast, in the amyloidogenic pathway, after cleavage by the β-secretase (BACE) to release sAPPβ, the remaining APP fragment (known as APP-CTFβ or C99) is then cleaved by the γ-secretase complex, liberating Aβ to the lumen and AICD to the cytosol. This latter pathway is likely favored in AD patients.

6Mammalian γ-secretase is a protease complex consisting of several components: presenilins 1 and 2 (PSEN1 and PSEN2), nicastrin (NCT), anterior pharynx defective (APH-1), and the presenilin enhancer (PEN-2;; ). PSEN1 and PSEN2 are the catalytic components of the γ-secretase complex. NCT works as a stabilizing cofactor required for γ-secretase complex assembly and trafficking and PEN-2 and APH-1 have a role in the maturation process of PSEN1 and PSEN2. Besides APP, the γ-secretase complex is also involved in the proteolysis of Notch receptors, and the first identification of any PEN-2 or APH-1 ortholog was in C. elegans as the result of a genetic screen for modifiers of the Notch pathway. Within the γ-secretase complex, only mutations in PSEN1 and PSEN2 have been associated with early onset AD.

7In C. elegans there is only one APP-related gene, apl-1. Like human APP, APL-1 contains a large extracellular region whose conserved E1 and E2 domains share 46 and 49% sequence similarity to human APP, respectively, a transmembrane domain, and a relatively small cytosolic domain, which shares 71% sequence similarity to human APP (Figure 1A; ). Notably, unlike APP but similar to APLP1 and APLP2, APL-1 does not contain the Aβ sequence.

8Two α-secretase proteins are present in C. elegans, SUP-17 and ADM-4. They work redundantly in the cleavage of the C. elegans Notch homologs, LIN-12 and GLP-1. However, no experiments thus far have tested whether SUP-17 or ADM-4 cleaves APL-1. No BACE ortholog has been identified by bioinformatic searches and no β-secretase activity that cleaves human APP has been detected in C. elegans, suggesting that APL-1 is only processed by the α/γ-secretase processing pathway. α-secretase cleavage of APL-1 releases the extracellular fragment, sAPL-1; subsequent cleavage of APL-1-CTFα by the γ-secretase complex liberates the intracellular domain (AICD; Figure 1B).

9The initial characterizations of human PSEN1 (then called S182) and PSEN2 (first named E5-1) described them as novel proteins with multiple transmembrane domains. The cellular functions of the presenilins were determined by their homology to the C. elegans protein, SEL-12. The two C. elegans Notch genes, lin-12 and glp-1, are involved in many cell fate decisions during development, including vulval cell specification and germline development sel-12/PSEN was identified in a genetic screen to isolate suppressors of a dominant Lin-12/Notch multivulva phenotype. Loss of sel-12/PSEN suppressed the Lin-12/Notch multivulva phenotype and produced a defect in egg laying that was rescued by introducing human PSEN1 or PSEN2, suggesting a conserved function between human and C. elegans presenilins. Like human PSENs, SEL-12/PSEN is cleaved to attain its final topology. A C. elegans PSEN gene family was identified and includes sel-12, hop-1, and spe-4; spe-4 is exclusively expressed in male gonadal cells and will not be further discussed. Knockdown of hop-1/PSEN in sel-12/PSEN mutants showed maternal effect lethality, germline defects, and missing anterior pharynx, defects associated with loss of glp-1/Notch function, suggesting that sel-12 and hop-1 function redundantly in the LIN-12 and GLP-1/Notch pathways. Similarly, mice carrying a null mutation in PSEN1 showed embryonic lethality, skeletal defects, and disrupted somite boundaries, similar to the phenotypes seen in Notch1 knockouts.

10In screening for novel mutants showing the glp-1/Notch phenotype of defective anterior pharynx,, ) identified two genes, aph-1 and aph-2, which encodes the C. elegans NCT ortholog. Independently, screened for enhancers of sel-12/PSEN activity and identified pen-2. aph-2/NCT, pen-2, and aph-1 are all required for proper Notch signaling. Human PSEN, NCT, Aph1α2, and PEN-2 were subsequently shown to physically associate and cooperatively regulate the maturation of individual components to form a proteolytically active γ-secretase complex.

11Like the mammalian APP family, apl-1 is expressed in multiple tissue types. apl-1 expression is observed in neurons, supporting cells, and head muscles throughout development, while expression in vulval muscles, vulval cells, and hypodermal seam cells is not detected until the L4 stage to adult.

12Inactivation of apl-1, such as with the yn10 null allele, results in a completely penetrant lethality during the first to second larval (L1–L2) transition due to a molting defect. apl-1 activity is also necessary for later larval transitions, as RNAi knockdown of apl-1 in an RNAi-sensitized background showed animals with molting defects during the L3–L4 and L4 to adult transitions. This lethality was rescued by microinjection of an apl-1 genomic fragment or cDNA. Hence, similar to the mammalian APP family, apl-1 has an essential function. High levels of apl-1 expression caused an incompletely penetrant L1 lethality (70% lethality), shortened body length, and morphogenetic, reproductive, and locomotory defects. These results indicate that levels of APL-1 must be tightly regulated as loss of APL-1 as well as high levels of APL-1 result in lethality. When sel-12/PSEN activity was reduced in transgenic animals with APL-1 overexpression, the 70% lethality was partially rescued, suggesting that SEL-12/PSEN regulates APL-1 cleavage and/or trafficking. The underlying basis of the loss- and gain-of-function apl-1 lethality is still unclear, but is not dependent on activation of CED-3/ caspase or necrotic cell death pathways. Characterization of apl-1 function may provide insights into the general function and pathways of APP, of which much is still unknown.

13The apl-1(yn5) mutant, which contains a deletion of the region encoding the APL-1 transmembrane and cytosolic domains, produces only the extracellular domain of APL-1 (APL-1EXT) and is viable. Because APL-1EXT is not further cleaved by α-secretase, APL-1EXT is slightly larger than sAPL-1 and is expressed at high levels in apl-1(yn5) mutants. Hence, the APL-1 extracellular domain is sufficient for viability, similar to the rescue of APP/APLP2 double mutants by the knock-in of sAPPα. However, although apl-1(yn5) mutants are viable, they display several phenotypes, including a slower developmental progression, decreased body length, reproductive defects, and temperature-sensitive lethality. Because these defects can be phenocopied by microinjection of APL-1EXT transgenes into wild-type animals, the phenotypes are due to overexpression of APL-1EXT and not due to loss of APL-1 signaling through its cytoplasmic domain. Interestingly, pan-neuronal expression of APL-1EXT, but not expression from muscle or hypodermal cells, is sufficient to rescue the lethality observed in apl-1 null mutants, suggesting that the cells (i.e., neurons) from which sAPL-1 is released as well as the extracellular milieu in which sAPL-1 travels is functionally relevant. We suggest that high levels of sAPP may also contribute to the pathology seen in AD patients. Down’s syndrome patients, whose chromosome 21 trisomy includes trisomy of APP, display a high incidence of AD and intellectual disability, perhaps contributed in small part by the high levels of APP expression.

14Decreasing apl-1 activity by RNAi resulted in hypersensitivity to aldicarb, an acetylcholinesterase inhibitor. Using apl-1 knockouts to test different apl-1 deletion constructs, determined that lack of sAPL-1 is responsible for the aldicarb hypersensitivity. These findings are consistent with mammalian studies, which show that a lack of APP and APLP2 impairs synaptic function at cholinergic neuromuscular junctions.

15Heterochronic genes, whose differential spatiotemporal expression ensures proper progression through larval stages and transition into adulthood, regulate expression of apl-1 in hypodermal seam cells. Loss of let-7 microRNA (miRNA) function caused precocious seam cell development and vulval bursting at the adult stage, leading to death. These let-7 phenotypes can be rescued by knockdown of apl-1. apl-1, however, is not a direct target of let-7 miRNA. NHR-25/Ftz-F1, which is a nuclear hormone receptor (NHR) that is required for completion of larval molts, binds an enhancer element in the promoter of apl-1 to regulate apl-1 expression in seam cells. nhr-25/Ftz-F1 transcripts are possible targets of the let-7 family of miRNAs for downregulation. Regulation of continued apl-1 expression in adult seam cells and other cell types is unknown.

16The apl-1(yn5) phenotypes require activity of the DAF-16/FOXO transcription factor, which is negatively regulated by the insulin pathway. C. elegans has only one insulin/IGF-1 receptor, DAF-2. Under favorable environmental conditions, such as when adequate food is present, signaling through the insulin pathway activates a conserved PI 3-kinase/AKT cascade, which causes phosphorylation of DAF-16/FOXO, thereby allowing reproductive development. Phosphorylation of DAF-16/FOXO causes its sequestration in the cytoplasm, thereby preventing it from entering the nucleus to activate its target genes, which regulate longevity, stress resistance, and dauer formation. Environmental conditions also affect other metabolic functions, such as reproductive behavior, which is inhibited under starvation conditions, and body size. Starvation survival behavior is regulated by DAF-16/FOXO activity and the insulin and DAF-7/TGFβ pathways work in parallel to regulate body length via daf-16/FOXO activity.

17The slowed development, decreased body size, and decreased reproductive rates of apl-1(yn5) mutants are dependent on daf-16/FOXO activity. At 20°C, mutants with decreased insulin signaling or apl-1(yn5) mutants showed a delayed developmental progression and shorter body length, which were enhanced when insulin signaling was decreased in apl-1(yn5) mutants [i.e., daf-2(e1370); apl-1(yn5) double mutants]; at 25°C, the apl-1(yn5) mutants with decreased insulin signaling went into L1 arrest. By contrast, when daf-16/FOXO activity was removed from apl-1(yn5) mutants, the delayed developmental progression, decreased reproductive rate, and smaller body length of apl-1(yn5) single mutants were suppressed. Furthermore, loss of daf-16/FOXO activity in apl-1(yn5) mutants with decreased insulin signaling rescued the short body length and L1 arrest phenotypes. These results suggest that sAPL-1 signals in a parallel pathway to the insulin pathway or modulates the DAF-2/insulin/IGF-1 pathway to activate daf-16/FOXO activity to affect developmental progression, reproductive rates, and body length (Figure 2). Mammalian sAPP may have similar roles in development.

18Activity of the daf-12/NHR signals in multiple pathways to integrate environmental stimuli with metabolic needs and can modulate the insulin pathway as well as function in an independent pathway. Decreasing daf-12/NHR activity in apl-1(yn5) mutants rescued the slow development, low reproductive rate, and decreased body length phenotypes. Hence, decreased insulin signaling and signaling through a parallel daf-12/NHR pathway converge to activate daf-16/FOXO for the phenotypes seen in apl-1(yn5) mutants. Noteworthy, levels of insulin/IGF-1 receptors are decreased in AD brains, and APP processing and Aβ production in vitro was modulated by insulin signaling. Analogous to C. elegans, sAPP may also act to modulate the insulin pathway.

19In transgenic mice expressing human or mouse APP, animals showed an increased lethality and learning defects that were not correlated with Aβ deposition; similarly, doubly transgenic mice carrying transgenes with APP and PSEN1 mutations showed learning defects that were not correlated with the number of Aβ plaques. The mechanisms underlying these defects are unclear. Use of the C. elegans model could be informative. C. elegans has many sensory modalities, including smell and taste. They respond to volatile and water soluble chemicals by moving toward or away from chemoattractive or chemorepulsive stimuli, respectively. Many chemoattractants and chemorepellants have been identified and the neural circuits mediating the chemosensory response identified. For instance, when C. elegans is given the choice between a neutral compound and a chemoattractant, such as benzaldehyde, animals will move toward benzaldehyde; this response is mediated by the AWC neurons; similarly, ASEL, a gustatory neuron, mediates chemoattraction to sodium acetate.

20Although apl-1 is not expressed in AWC neurons and the morphology of sensory neurons appears wild type with GFP markers, the overall chemoattractive response to benzaldyhyde and sodium acetate was decreased in apl-1(yn10/+) heterozygotes and transgenic animals that overexpress APL-1 [e.g., ynIs79 (Papl-1::apl-1::GFP)]. The chemotaxis response was restored in APL-1 overexpression lines [e.g., ynIs79 (Papl-1::apl-1::GFP)] when insulin signaling was decreased, but not when daf-16/FOXO activity was decreased, suggesting that daf-16/FOXO activity is needed for normal chemotaxis in transgenic lines overexpressing APL-1 (Figure 2). Pan-neuronal expression of APL-1 or targeted overexpression of APL-1 in the AWC or ASEL neurons resulted in wild-type chemotaxis responses. By contrast, ectopic expression of apl-1 with the snb-1 promoter, which drives pan-neuronal and multi-cell type expression, resulted in no chemotaxis response to benzaldehyde or sodium acetate. When signaling through the DAF-2/insulin/IGF-1 receptor, DAF-12/NHR, or DAF-7/TGFβ was decreased, the chemotaxis response toward benzaldehyde and sodium acetate in these transgenic lines was restored, indicating that the loss of the chemotaxis response due to ectopic apl-1 signaling in cells outside the nervous system is dependent on insulin and TGFβ signaling.

21In addition to chemosensory responses, C. elegans is also capable of associative chemosensory plasticity. For example, when benzaldehyde was paired with starvation for as short as 30 min, C. elegans showed a significant reduction in preference for benzaldehyde; persistence of this plasticity was positively correlated with the length of pairing time, suggestive of stable memory formation. Both chemotaxis and associative plasticity are dependent on insulin signaling. Little associative plasticity, however, was observed after pairing benzaldehyde with starvation for 60 min in animals with pan-neuronal APL-1 expression. The plasticity could be restored when daf-16/FOXO, daf-12/NHR, or daf-7/TGFβ activity was decreased, indicating that the impaired associative plasticity with pan-neuronal APL-1 expression requires daf-16/FOXO, daf-12/NHR, and daf-7/TGFβ activity.

22Touch habituation is another sensory characteristic affected by pan-neuronal APL-1 expression. When a gentle touch is applied to the head of the animal, C. elegans responds by moving backward; conversely, when touched on the tail, the animal moves forward. This response to gentle body touch is mediated by six mechanosensory touch neurons. After six cycles of alternating head/tail touches, wild-type animals habituated and became unresponsive. apl-1(yn10/+) heterozygotes or transgenic animals that overexpress APL-1 showed touch habituation. By contrast, animals with pan-neuronal APL-1 expression were slow to habituate and required more alternating head/tail touch cycles before becoming habituated. Collectively, these results indicate that pan-neuronal overexpression of APL-1 causes learning defects. These results parallel those seen in mammalian models in which overexpression of APP leads to cognitive defects, independent of Aβ aggregates, thereby suggesting that sAPP activity, in addition to Aβ aggregates, contributes to cognitive defects. Whether these cognitive defects depend on the DAF-16/FOXO transcription factor and/or TGFβ signaling remains to be tested.

23APL-1, like APP, is transported from the cell body to synapses. UNC-108, which is a neuronally expressed GTPase and localizes to the Golgi complex and early endosomes, is involved in the maturation of dense core vesicles and the packaging of APL-1 into mature dense core vesicles. Both UNC-116/kinesin-1 and UNC-104/KIF1A/kinesin-3 are involved in the anterograde transport of APL-1, but only UNC-116/kinesin-1 and dynein motors are responsible for the retrograde transport of APL-1 back to the cell body. The rates of anterograde and retrograde transport of APL-1 vesicles are 1.1 μm/s and 1.6 μm/s, respectively. Hence, APL-1 is transported similarly as in mammalian models where kinesin-1 is responsible for APP axonal transport. Surprisingly, mutations in unc-116/kinesin-1 and unc-104/KIF1A/kinesin-3 both caused decreased levels of APL-1 expression without affecting transcript levels, suggesting that without transport motors, APL-1 does not accumulate in cell bodies because of protein degradation. APL-1 is also internalized from the cell surface of neurons via a RAB-5-dependent endocytosis.

24The Fe65 family of proteins binds the cytoplasmic YENPTY sequence of APP, APLP1, and APLP2, via their PTB2 domain. Likewise, the sole family member ortholog in C. elegans, FEH-1, has a WW domain and PTB1 and PTB2 domains, which closely resemble those of the Fe65 family, and the PTB2 domain of FEH-1 interacts with APL-1.

25FEH-1 is expressed in pharyngeal muscle and neuronal processes and is necessary for survival. Inactivation of feh-1 caused an incompletely penetrant embryonic lethality. Survivors showed little pharyngeal pumping and were unable to feed, thereby resulting in L1 arrest. Decreasing feh-1 activity or decreasing feh-1 dosage caused pharyngeal pumping rates to increase, suggesting that the rate of pharyngeal pumping is feh-1 dosage dependent. However, the functional significance of FEH-1 and APL-1 interactions is unclear as apl-1(yn5) mutants, which do not have an AICD domain, and apl-1(yn10/+) heterozygotes do not have defective pumping rates.

26Aβ peptide, the cleavage product of APP believed to underlie the pathology of AD, is not present in APL-1 nor does C. elegans possess β-secretase activity to produce Aβ. Nevertheless, C. elegans provides a powerful in vivo genetic system to study the effects of neurotoxic Aβ through transgene analysis. Many transgenic strains have been generated in which a signal sequence followed by the human Aβ sequence is expressed in all cells, in all neurons, in specific subsets of neurons, or in muscle cells (Figure 4). These strains produce either Aβ1-42 or Aβ3-42.

27AD is a late onset neurodegenerative disease. C. elegans expressing human Aβ3-42 in muscle tissue showed an age-dependent paralysis at 20°C; paralysis occurred more rapidly and more severely when Aβ1-42 was produced at 25°C. The level of muscle paralysis was significantly decreased when insulin signaling was decreased. Furthermore, inhibiting daf-16/FOXO and hsf-1, which encodes a heat shock protein transcription factor, reversed the effects of decreased insulin signaling. Hence, the paralysis effects of Aβ correlates with age and is dependent on insulin signaling.

28Since aggregated Aβ is toxic to neurons and causes muscle paralysis in C. elegans, molecules and pathways that can prevent the formation or promote the disassembly of Aβ aggregates can be screened for in C. elegans. For instance, when C. elegans extracts are incubated with aggregated human Aβ3-42 in the presence or absence of protease inhibitors, disaggregation occurred, but disaggregation did not occur when extracts were either heated to denature proteins or incubated with proteinases. Hence, an unidentified protein or protein complex in C. elegans extracts can disaggregate Aβ3-42 aggregates.

29Several orthologs to human heat shock (HSP) chaperone proteins were found to interact directly with Aβ3-42 in C. elegans. C. elegans HSP-16 proteins, HSP-16-1, HSP-16-2, and HSP-16–48, orthologs of αB-crystallin, bound intracellular Aβ3-42 monomers and soluble Aβ3-42 oligomers, but not fibrillar Aβ3-42. Moreover, hsp-16 transcript levels were upregulated in Aβ3-42 transgenic lines, but whether these chaperone proteins protect against or promote Aβ paralysis is unclear. By contrast, increased expression of the HSP70 chaperones had a protective role by suppressing paralysis. These results are consistent with human studies showing that HSP70 and αB-crystallin were upregulated in AD brains and binds Aβ.

30Transgenic lines in which Aβ is expressed in glutamatergic neurons showed age-dependent neurodegeneration, whereby 7-day adults showed 75% glutamatergic neurodegeneration. This degeneration was suppressed when genes involved in clathrin-mediated endocytosis, such as unc-11, unc-26, Y44E3A.4, C. elegans RTS1 ortholog, C. elegans ADE12 ortholog, and human CRMI, were co-expressed with Aβ, and enhanced when a PBS2/MAP2K4 mitogen-activated protein kinase transgene was co-expressed with Aβ in glutamatergic neurons. Interestingly, mutations in the C. elegans human REST ortholog spr-4, which suppressed the sel-12/PSEN egg-laying defect, also enhanced the degeneration seen in the transgenic animals expressing Aβ in glutamatergic neurons. Modifying clathrin-mediated endocytosis in rat cortical neurons was similarly neuroprotective against Aβ aggregates. In addition, early stage AD brains showed higher expression of REST target genes, while late stage AD and frontotemporal dementia (FTD) brains showed lower expression. Hence, REST may confer neuroprotective benefits in C. elegans and in humans.

31The C. elegans Aβ model also proves useful in screens to identify drugs that disaggregate Aβ. The drug PBT2, an 8-hydroxy quinoline analog, reversed AD phenotypes in mice within days. Similarly, C. elegans expressing inducible Aβ1-42, which become paralyzed within 48 h after induction, were protected against paralysis when exposed to PBT2.

32In mammals, the LDL receptor family is responsible for many functions, including binding ligands for internalization and degradation and cholesterol metabolism. Binding of LRP1 to sAPP770 or full-length APP770, one of the APP isoforms (Figure 1), causes its internalization and degradation; disrupting cell surface APP internalization with an LRP-antagonist increases sAPPα processing and full-length APP at the cell surface and decreases Aβ formation, suggesting that LRP1-APP interactions favor APP processing through the amyloidogenic pathway. Apolipoprotein E and LRP2/megalin have also been implicated in Aβ clearance.

33C. elegans LRP-1 most closely resembles mammalian LRP2/megalin. C. elegans does not have the ability to synthesize cholesterol and, therefore, must rely on dietary sources. Inactivation of lrp-1 resulted in late larval lethality due to molting defects during the L3–L4 transition. When wild-type C. elegans were grown in the absence of cholesterol, the molting defects of the lrp-1 knockouts were phenocopied, suggesting that LRP-1 is involved in cholesterol uptake from the environment. LRP-1 is expressed in the epithelial hypodermal cells, hyp6 and hyp7, where it localizes to their apical surface and where apl-1 is also expressed in adults. Similarly, its mammalian counterpart, LRP2/megalin, is mainly expressed at the apical surface of epithelial cells.

34LRP2/megalin interacts with different domains of APP. A physical interaction between APL-1 and LRP-1 has not been determined. Expression of LRP-1 with an N-terminal domain truncation did not rescue the lethality of apl-1 null mutants, suggesting that sAPL-1 is not activating an lrp-1 pathway. When lrp-1 expression is decreased or when wild-type animals are deprived of dietary cholesterol, neurotransmission is affected. However, apl-1 null mutants die at an earlier stage in development than lrp-1 null mutants, suggesting that apl-1 functions in earlier developmental pathways that are necessary for survival.

35Accumulation of neurofibrillary tangles in cell bodies is another hallmark characteristic of AD and other neurodegenerative disorders. The major component of these tangles is tau, which belongs to the family of microtubule-associated proteins (MAPs) that includes MAP2 and MAP4. MAPs share characteristic homology domains, including a proline-rich domain and a region of a variable number of tandem amino acid repeats (Figure 3A;,,;;; ). Tau is the predominant MAP expressed in axons, while MAP2 is expressed in dendrites and MAP4 is expressed in dividing cells. MAPs bind microtubules and are responsible for promoting microtubule assembly and stability (reviewed in ). MAP family members appear to have redundant functions; mice in which tau was knocked out were viable, but showed increased levels of MAP1A, suggesting that upregulation of MAP1A can compensate for the lack of tau in vivo. Tau phosphorylation affects its ability to bind microtubules and can cause a conformational change that favors tubulin assembly (Figure 3B; ). Aberrant hyperphosphorylation of tau, however, impairs its ability to bind microtubules, thus resulting in their disassembly. In addition, phosphorylated tau self-aggregates into PHFs and presumably generates the intracellular neurofibrillary tangles characteristic of AD patients (Figure 3B;;,,; ).

36Because of the functional redundancy of MAPs, their specific functions have been difficult to determine. C. elegans has only one tau homolog protein with tau-like repeats 1 (PTL-1;; ). PTL-1 exists as two isoforms, PTL-1A and PTL-1B, with five or four tandem repeats, respectively (; Figure 3A). They have a high level of sequence homology with mammalian tau, especially in the C-terminal microtubule binding region. Both PTL-1A and PTL-1B bound microtubules in vitro and induced tubulin polymerization. PTL-1 is initially expressed embryonically in the epidermis of elongating embryos and in head neurons; in larval and adult animals PTL-1 is expressed mainly in the mechanosensory neurons mediating gentle body touch (Figure 3B;; ), although transcriptional fusions show a wider expression pattern in neurons and stomatointestinal cells.

37Loss of ptl-1/tau results in an incompletely penetrant lethality at the same stage of embryogenesis at which PTL-1/tau expression is first observed. ptl-1/tau mutants that escaped lethality showed normal development, but had a shortened lifespan, and although the overall integrity of microtubule structure appeared unaffected at the light microscopic level, there was a significant reduction in gentle touch sensitivity as compared to wild-type. These touch defects were enhanced in mutants with defects in β- and α-tubulin, indicating that the absence of full-length PTL-1/tau disrupts mechanosensation, but it does so independently of tubulin. In mutants in which only the C-terminal microtubule binding repeats of PTL-1/tau are deleted, touch sensitivity was identical to that of wild-type, suggesting that the N-terminal domain of PTL-1/tau is sufficient for gentle touch responses.

38As C. elegans ages, mechanosensory touch neurons exhibit age-related morphological changes: cell bodies initially elaborate branches and axons subsequently display blebbing and branching. Strikingly, mechanosensory touch neurons in ptl-1/tau mutants displayed these aging characteristics at higher incidences and at an earlier stage than wild-type animals; GABAergic neurons also showed age-related phenotypes, such as ectopic branching. Expression of a human tau isoform (htau40) in ptl-1/tau mutants rescued the touch insensitivity, but not the morphological aging defects, indicating that htau40 shares some functional conservation with PTL-1/tau. Interestingly, when ptl-1/tau was expressed in Sf9 cells, cells projected neurite-like processes that were positive for PTL-1/tau immunoreactivity and that were indistinguishable from those visualized when htau40 was expressed in Sf9 cells. Although wild-type PTL-1/tau has not been reported to aggregate into fibrils, PTL-1/tau, like human tau, clearly has an essential role in maintaining neuronal integrity, controlling neuronal aging, and affecting lifespan. While there are no known mutations in tau that are associated with AD, tau mutations are associated with FTD with parkinsonism (FTPD-17), another form of dementia (;;; see Section “Frontotemporal Dementia”).

39Frontotemporal dementia (FTD) is a group of neurodegenerative disorders characterized by severe brain frontotemporal lobar degeneration (reviewed in ). In some cases it may be hard to distinguish between FTD and AD; however, FTD usually develops earlier in life and is more likely to have a genetic component. Many mutations can cause FTD with or without motor neuron disease. Two mutations have been well characterized and are associated with specific types of FTD: tau-positive FTD linked to chromosome 17 (FTD-17) and FTD caused by TDP43 proteinopathy (FTD-TDP43). Patients with FTD-17 suffer behavioral changes and often Parkinson-like motor problems. While mutations in the tau gene MAPT are not described in familial or sporadic AD, MAPT tau mutations are linked with FTD-17.

40Several transgenic lines expressing human tau harboring FTD-17 mutations (htau-FTD-17) have been generated in C. elegans (Figure 4B) (see also Section “PTL-1 as a Tau Model”;;;; ). Pan-neuronal transgene expression of wild type or htau-FTD-17 caused an uncoordinated phenotype that progressively worsened with age, an accumulation of insoluble tau, and neurodegeneration. Similarly, expression of htau-FTD-17 in touch neurons resulted in a decrease in the touch response due to neuritic abnormalities and tau accumulation. Using these C. elegans models of tauopathy in forward genetic screens, Kraemer and co-workers identified two new factors, SUT-1 and SUT-2, that may participate in the pathological pathway activated by tau. Moreover, down-regulation of the human SUT-2 homolog (MSUT-2) in mammalian cell lines caused a marked decrease in tau aggregation, suggesting that MSUT-2 may be a good candidate target for FTD therapies. More recently, generated two htau-FTD-17 transgenic models: one with a pro-aggregant mutated form of human tau (deletion of K280) and a second with mutated forms of human tau (I277P and I308P) that prevented tau aggregation. The tau (ΔK280) transgenic line had high levels of tau aggregation, which caused uncoordinated movement in adults, axonal defects, and alterations in presynaptic structures; the locomotory defects could be partially suppressed by a compound of the aminothioenopyridazine (ATPZ) class cmp16, suggesting that this compound may be neuroprotective. The tau (I277P and I308P) transgenic lines had low levels of tau aggregates and displayed only mild phenotypes with significantly less morphological abnormalities.

41Accumulation of TDP-43 [transactive response (TAR) DNA-binding protein] is found in ∼50% of the cases of FTD and has numerous genetic causes. However, only one case has been reported with mutations in the TDP-43 gene. C. elegans models overexpressing human TDP-43 or its C. elegans ortholog TDP-1 recapitulates some of the FTD phenotypes, including neurotoxicity and protein aggregation (see also Section “Amyotrophic Lateral Sclerosis”).

42C9orf72 encodes a protein that regulates endosomal trafficking and autophagy in primary neurons and neuronal cells. It is expressed in multiple tissue types, including cerebellar cortex and spinal cord. Hexanucleotide (GGGGCC) repeat expansions in a non-coding region of C9orf72 are found in patients with amyotrophic lateral sclerosis (ALS) and FTD, providing the first genetic link between the two diseases, although it remains unclear how C9orf72 hexanucleotide expansion triggers ALS and FTD pathology. Mutations in the C. elegans C9ORF72 ortholog alfa-1 caused age-dependent motility defects, leading to paralysis and degeneration of GABAergic motoneurons, suggesting that a loss-of-function mechanism is involved in the C9ORF72-dependent pathogenesis.

43Parkinson’s disease (PD) is a progressive neurodegenerative disorder that affects the control of body movements. The impaired motor control in PD is the result of the death of dopaminergic (DA) neurons. The disease is characterized by the accumulation of α-synuclein into neuronal inclusions called Lewy bodies. Most PD cases are of unknown cause. However, ∼5–10% of PD cases are familial and include mutations in the following genes: α-synuclein, parkin (PRKN), leucine-rich repeat kinase 2 (LRRK2), PTEN-induced putative kinase 1 (PINK1), and ATP13A2.

44α-synuclein is a presynaptic neuronal protein whose cellular function is not well understood, but may include controlling the supply of synaptic vesicles in neuronal terminals and regulating dopamine release. It is a small acidic protein (14 kDa) whose sequence can be divided into three domains: the N-terminal α-helical domain (amino acids 1–65), the central hydrophobic domain (residues 66–95), and the acidic carboxyl-terminal domain (residues 96–140; ). Three mutations in the α-helical domain (A53T, A30P, E46K) are linked with autosomal dominant early onset PD, suggesting that these mutations can predispose to oligomer and fibril formation. Because C. elegans has no α-synuclein ortholog, C. elegans models are based on transgenic worms overexpressing wild-type or mutant forms of human α-synuclein (Figure 4B). Although different transgenic lines showed some differences, most lines with pan-neuronal or DA neuronal expression of wild-type or mutated α-synuclein (A53T and/or A30P mutations) displayed locomotory defects and degeneration of dopamine neurons. Furthermore, downregulating the activity of the nuclease EndoG decreased α-synuclein toxicity in DA neurons. EndoG is a mitochondria-specific endonuclease that mediates cellular death by apoptosis. When cell death is induced, EndoG translocates from the mitochondria to the nucleus to fragment DNA. Approximately 50% of the dopamine neurons expressing α-synuclein degenerate, whereas a mutation in cps-6, which encodes the C. elegans EndoG ortholog, rescues this degeneration. Similar results were found in yeast, flies, and human cells suggesting that EndoG is a conserved requirement for α-synuclein toxicity.

45C. elegans models of α-synuclein overexpression-induced toxicity have also been examined by whole genome RNAi knockdown and microarray screenings. These screens highlighted the importance of endocytosis for ameliorating α-synuclein-dependent neurotoxicity. Transgenic lines expressing α-synuclein specifically in body wall muscle cells produced inclusions as animals aged, resembling a feature of neurons in patients with PD; the number of inclusions decreased when genes affecting different biological processes, such as vesicle and lysosomal trafficking (W08D2.5/ATP13A2), lipid metabolism, and lifespan control (sir-2.1,lagr-1), were knocked down. In a genome-wide microarray analysis to identify genes that were modulated in C. elegans overexpressing wild-type or A53T human α-synuclein, seven genes encoding components of the ubiquitin-proteasome machinery and 35 mitochondrial function genes were found to be upregulated, while nine genes encoding histones H1, H2B, and H4 were down regulated. These data provide support for the role of the proteasome complex and mitochondrial proteins in mediating neurotoxicity.

46Parkin (human PARK2) is a component of an ubiquitin E3 ligase that is part of the proteasome complex. Mutations in PARK2 have been associated with early onset recessive forms of PD. C. elegans has a parkin ortholog, PDR-1. A truncated form of PDR-1(Δaa24–247) encoded by the in-frame deletion null allele pdr-1(lg103) had altered solubility and propensity to aggregate when expressed in cell lines, resembling parkin mutant proteins in PD. Furthermore, pdr-1 mutants were hypersensitive to different proteotoxic stress conditions, suggesting that PDR-1/PARK2 mutations act to block the proteostasis machinery, thereby making it easier for proteins to abnormally fold and aggregate.

47Mutations in LRRK2/leucine-rich repeat kinase 2 are the most common known cause of late-onset PD. LRRK2 belongs to the LRRK family; gain-of-function LRRK2 mutations interfere with chaperone-mediated autophagic functions and presumably decrease levels of α-synuclein degradation. Transgenic worms overexpressing pathogenic mutant forms of LRRK2 in DA neurons caused DA neurodegeneration. Interestingly, treatment of the transgenic worms with kinase inhibitors resulted in arrested neurodegeneration, suggesting that LRRK2 kinase activity is important for its pathogenesis.

48Many studies have reported a link between toxin exposure and increased risk of PD. C. elegans has been used to test different toxins and help elucidate the mechanism by which they produce neurotoxicity. Administration of the 6-hydroxydopamine (6-OHDA) neurotoxin to C. elegans produced specific degeneration of dopamine neurons. By performing forward genetic and high-throughput chemical screens, mutations within the dopamine transporter dat-1 were found to suppress 6-OHDA sensitivity and bromocriptine, quinpirole, and acetaminophen, and plant extracts from Bacopa monnieri and Uncaria tomentosa were found to be neuroprotective. These data demonstrate that pathological characteristics of PD can be recapitulated in C. elegans models and used to investigate the mechanism by which α-synuclein and other PD proteins produce neurotoxicity and cause motor defects.

49Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by the death of motor neurons in brain and spinal cord and progressive paralysis of the body. Approximately 10% of ALS cases are familial and associated with mutations in several genes. The most common mutation in familial ALS is found in the superoxide dismutase enzyme (SOD1) with more than 160 different mutations identified. SOD1 is a ubiquitously expressed protein that converts the toxic radical superoxide anion to hydrogen peroxide. Although it is not clear yet how SOD1 mutations causes motor neuron degeneration, toxicity is likely generated by a gain-of-function mechanism and associated with misfolding and aggregation of the enzyme.

50Transgenic lines expressing mutant human SOD1 proteins have been successfully generated in C. elegans and recapitulate the motor neuron degeneration and paralysis characteristic of ALS patients (Figure 4B). The locomotion defect caused by pan-neuronal expression of the SOD1(G85R) mutant isoform was reduced when insulin signaling was decreased, suggesting that decreased insulin signaling increases the capacity of cells to prevent the accumulation of toxic non-soluble proteins and opening the possibility of finding new therapeutic targets. Similarly, when wild-type or mutant SOD1(G93A) was expressed exclusively in GABAergic motor neurons, animals showed an age-dependent paralysis and accumulation of wild-type and mutant SOD1(G93A), although defects were more severe in the mutant lines; interestingly, the SOD1 aggregates were soluble in the wild-type SOD1 lines and insoluble in the SOD1(G93A) lines. In addition, motor neurons showed axonal guidance defects during development and caspase-independent cell death in adulthood in the wild-type and SOD1(G93A) lines.

51Other genes associated with ALS have also been modeled using C. elegans. TDP-43 [TAR DNA-binding protein] is a 43 kDa RNA binding protein identified as the main component of ubiquitinated protein aggregates found in patients with sporadic ALS and also in some cases of FTD (see Section “Frontotemporal Dementia”). TDP-43 is normally located in the nucleus of neurons, but dominant mutations in TDP-43 cause aberrant localization of TDP-43 in the cytoplasm, thereby preventing it from functioning in the nucleus. C. elegans has one TDP-43 ortholog, TDP-1. TDP-1 controls longevity and oxidative stress in the worm by regulating the insulin pathway. Overexpression of tdp-1/TDP-43 resulted in toxicity and decreased lifespan, analogous to the phenotypes found in ALS patients. In transgenic worms expressing TDP-43 harboring ALS-associated mutations, proteotoxicity affecting neuronal functions was induced. Similar results were found when the RNA binding protein FUS with ALS-related mutations was expressed in the nematode.

52Excess exposure to some pesticides and chemicals, such as the metalloid selenium, have been implicated in the etiology of ALS. Exposure to high levels of sodium selenite in the worm induced neurodegeneration and resulted in paralysis. When insulin pathway activity was reduced, the adverse effects of environmental selenium exposure was altered. Overall, the C. elegans models have highlighted the possible importance of the insulin and autophagy pathways in the generation of ALS.

53Huntington’s disease (HD) is a progressive neurodegenerative disorder inherited through autosomal dominant mutations of the IT15 gene. ITI5 encodes the huntingtin protein, whose functions remain unknown. The mutations result in an N-terminal polyglutamine (polyQ) expansion. In normal individuals, up to 34 repeats have been reported, whereas in HD afflicted individuals, up to 100 polyQ repeats have been recorded. The huntingtin-polyQ (HdhQ) proteins form aggregates, whose toxicity is determined by the length of the polyQ expansion and which cause swollen, disorganized, and ribosome-deficient endoplasmic reticulum and chromatin irregularities. Eventually, cellular defects caused by the aggregates culminate in HD symptoms, which include involuntary movement, cognitive impairment, and loss of neurons in the striatum and deep layers of the frontal cortex.

54Although C. elegans does not have a huntingtin homolog, transgenic C. elegans models that express an N-terminal human huntingtin (htt) fragment with different numbers of CAG repeats have been used to model HD and identify genes that prevent polyQ aggregates (Figure 4B). The models generally express the repeats in specific neurons, such as the ASH sensory neurons, which are multi-modal sensory neurons that mediate avoidance to chemo- and mechanosensory stimuli. In transgenic animals expressing htt171 with 150 CAG repeats (htt171-Q150), 13% of the ASH neurons began to lose function after 8 days, suggesting an age-dependent degeneration. This loss of ASH function was reversed in a ced-3/caspase or hda-3/HDAC mutant background, suggesting that processes characteristic of apoptotic cell death and histone deacetylases play a role in HD. By contrast, the number of htt171-Q150 aggregates and neurodegeneration were enhanced when genes mediating autophagy, CREB, CREB binding proteins, and pqe-1 were disrupted, suggesting that autophagy and activation of CREB target genes decreases htt171-Q150 aggregation and are neuroprotective. Transgenic animals expressing fewer CAG repeats (2, 23, and 95 polyQ) showed normal ASH function. The onset of behavioral defects are consistent with most cases of HD, in which symptoms usually appear during midlife and fewer than 10% of reported cases occur before the age of 21.

55In a different HD model, htt57-Q128 was expressed in the touch mechanosensory neurons. These transgenic animals did not show neurodegeneration, but had a significantly reduced response to posterior touch and a milder defect in anterior touch response. The touch neurons contained polyQ aggregates and morphological abnormalities primarily along axonal processes. The touch insensitivity could be rescued by activating Sir2 sirtuins, which act through the DAF-16/FOXO transcription factor to promote longevity. Similarly, in neuronal cell lines derived from knockin HdhQ111 mice, activation of sirtuins reduced the level of cell death. Additionally, in an RNAi based screen for genes that suppressed htt57-Q128 defects, identified C. elegans genes were also upregulated in the striatum of mouse HD models. Thus, C. elegans is a useful model to identify additional genes that may protect against or contribute to defects caused by polyQ expansions.

56RNAi knockdown of dnj-27/ERdj5, an ER luminal protein upregulated in response to ER stress, exacerbated the impaired mobility observed when a Q40 transgene is expressed in body wall muscles, suggesting that dnj-27 interacts with polyQ and protects against polyQ induced paralysis.

57C. elegans has the advantage that it is transparent, allowing visualization of the formation of that aggregates, including aggregates made by shorter polyQ tracts, whereas only longer tracts are visible in mammals. To determine the threshold number of polyQ repeats needed to elicit a morphological and behavioral response, varying lengths of polyQ repeats were tested in C. elegans. Pan-neuronal expression of more than 40 polyQ led to variable protein aggregation and paralysis. These data suggest that 40 polyQ may be the critical number of repeats to elicit HD symptoms and are consistent with unaffected humans who have up to 34 polyQ repeats and HD patients who have as few as 42 repeats.

58Overall, C. elegans HD models illustrate that human huntingtin polyQs disrupt the morphology and function of sensory neurons. The genetic and RNAi screens highlight candidate genes that may be involved in HD pathogenesis in mammalian models and provide insights into genes that may serve a protective role against polyQ toxicity. In addition to HD, other diseases caused by polyQ repeats include spinocerebellar ataxias and spinal and bulbar muscular atrophy. Hence, using C. elegans provides another approach toward determining how polyQ pathogenicity contributes to neurodegeneration.

59The use of C. elegans to study AD and other neurodegenerative diseases has, as many other models, many advantages as well as some drawbacks. Major advantages of C. elegans include the ability to perform forward genetic, RNAi, and high throughput chemical screens and the ease of generating transgenic lines. These benefits have been effective in informing the role of APP and the presenilins and identifying components of the γ-secretase complex. The function of APP and the pathways in which it acts are still unclear. C. elegans presents a complementary system to understand the function and pathways of an APP-related protein, APL-1. Furthermore, overexpression of APL-1 by mutation or by transgene induces phenotypes that converge on the insulin/DAF-16/FOXO pathways, similar to what has been found in mammals. Although APL-1 does not contain the Aβ sequence and C. elegans does not have β-secretase activity, transgenic lines that produce Aβ expression pan-neuronally or in muscle are being used to identify pathways that detoxify the Aβ aggregates, some of which also involve the insulin/DAF-16/FOXO pathways. Whether these models are relevant to human pathology or whether the pathways will be conserved in humans are unknown; however, they present alternative approaches to understanding neurodegenerative diseases for which there are currently few effective therapies. Human tau, as well as mutant tau isoforms, have also been expressed in the worm to recapitulate AD and FTD phenotypes. Recent findings have shown that PTL-1 regulates neuronal aging in the worm. These findings may be important to link aging and tau pathology in AD and FTD patients. Although C. elegans transgene models have many advantages, they also have several disadvantages. In C. elegans, transgenes are present as extrachromosomal arrays and are not integrated into the genome as they are in other systems; a few copies to several hundred copies of the transgene are present in the arrays, so the level of overexpression can be much higher than what is found in vivo. Fortunately, methods for single copy insertions have now been developed.

60AD is considered a multifactorial disease in which other risk factors, such as neuroinflammation, head trauma, and diabetes, may be important in the development of the disease. The C. elegans nervous system is simple compared to the human nervous system. This simplicity allows researchers to study neuronal function and neural circuits in a tractable system. However, the complex network of connections and cell interactions found in humans is not mimicked in C. elegans and this complexity may underlie some of the pathology of neurodegenerative diseases. Nevertheless, most of the pathways and signaling molecules in C. elegans are conserved between worms and mammals. The goal is to translate some of the C. elegans insights into understanding the pathology of AD and other neurodegenerative diseases and designing effective strategies to treat the diseases.

61The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.